1. Field of the Invention
The invention is directed to purified and isolated novel ss3939 polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.
2. Description of Related Art
Of recent interest are sugar-binding proteins, known as lectins, which mediate both pathogen recognition and cell-cell interactions using structurally related calcium-dependent carbohydrate-recognition domains, or C-type lectin domains (Drickamer, K. J., Biol. Chem. 263:9557–9560, 1988). In recognizing pathogens, certain C-lectin-containing proteins, such as the macrophage mannose receptor, bind terminal monosaccharide residues characteristic of fungal and bacterial cell surfaces (Fraser, I. P., Semin. Immunol., 10(5):363–72, 1988). The macrophage mannose receptor contains seven tandemly repeated C-type lectin domains, each of which consists of about 110 to 130 residues. There are four cysteines which are perfectly conserved and involved in two disulfide bonds.
The conserved size and amino acid composition of the C-type lectin domain provides a template for computer-based sequence comparison in the identification of novel sequences containing C-type lectin domains. Novel sequences encoding C-type lectin domains or domains similar to C-type lectin domains may have functions similar to those ascribed to previously described C-type lectin domain-containing molecules, such as recognizing, binding, and mediating the uptake of pathogens. The identification of novel molecules containing domains similar to the C-type lectin domain may thus lead to improved therapies for enhancing cellular immunity.
In addition, in view of the continuing interest in antigen recognition and the immune system, there is still a need in the art for the identity and function of proteins involved in cellular and immune responses.
In another aspect, the identification of the primary structure, or sequence, of an unknown protein is the culmination of an arduous process of experimentation. In order to identify an unknown protein, the investigator can rely upon a comparison of the unknown protein to known peptides using a variety of techniques known to those skilled in the art. For instance, proteins are routinely analyzed using techniques such as electrophoresis, sedimentation, chromatography, sequencing and mass spectrometry.
In particular, comparison of an unknown protein to polypeptides of known molecular weight allows a determination of the apparent molecular weight of the unknown protein (T. D. Brock and M. T. Madigan, Biology of Microorganisms, pp. 76–77, Prentice Hall, 6th ed., 1991). Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein (New England Biolabs Inc. Catalog:130–131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758). However, the molecular weight standards may not correspond closely enough in size to the unknown protein to allow an accurate estimation of apparent molecular weight. The difficulty in estimation of molecular weight is compounded in the case of proteins that are subjected to fragmentation by chemical or enzymatic means, modified by post-translational modification or processing, and/or associated with other proteins in non-covalent complexes.
In addition, the unique nature of the composition of a protein with regard to its specific amino acid constituents results in unique positioning of cleavage sites within the protein. Specific fragmentation of a protein by chemical or enzymatic cleavage results in a unique “peptide fingerprint” (D. W. Cleveland et al., J. Biol. Chem. 252:1102–1106, 1977; M. Brown et al., J. Gen. Virol. 50:309–316, 1980). Consequently, cleavage at specific sites results in reproducible fragmentation of a given protein into peptides of precise molecular weights. Furthermore, these peptides possess unique charge characteristics that determine the isoelectric pH of the peptide. These unique characteristics can be exploited using a variety of electrophoretic and other techniques (T. D. Brock and M. T. Madigan, Biology of Microorganisms, pp. 76–77, Prentice Hall, 6th ed. 1991).
Fragmentation of proteins is further employed for amino acid composition analysis and protein sequencing (P. Matsudiara, J. Biol. Chem. 262:10035–10038, 1987; C. Eckerskorn et al., Electrophoresis, 9:830–838, 1988), particularly the production of fragments from proteins with a “blocked” N-terminus. In addition, fragmented proteins can be used for immunization, for affinity selection (R. A. Brown, U.S. Pat. No. 5,151,412), for determination of modification sites (e.g. phosphorylation), for generation of active biological compounds (T. D. Brock and M. T. Madigan, Biology of Microorganisms, pp. 300–301, Prentice Hall, 6th ed. 1991), and for differentiation of homologous proteins (M. Brown et al., J. Gen. Virol. 50:309–316, 1980).
In addition, when a peptide fingerprint of an unknown protein is obtained, it can be compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011–5015, 1993; D. Fenyo et al., Electrophoresis 19:998–1005, 1998). A variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (Internet site: prospector.uscf.edu), MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site: www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form.html), and ProFound (Internet site: www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance. The programs compare these molecular weights to protein molecular weight information stored in databases to assist in determining the identity of the unknown protein. Accurate information concerning the number of fragmented peptides and the precise molecular weight of those peptides is required for accurate identification. Therefore, increasing the accuracy in determining the number of fragmented peptides and their molecular weight should result in enhanced likelihood of success in the identification of unknown proteins.
In addition, peptide digests of unknown proteins can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J. K. Eng et al., J. Am. Soc. Mass Spec. 5:976–989, 1994; M. Mann et al., Anal. Chem. 66:4390–4399, 1994; J. A. Taylor et al., Rapid Comm. Mass Spec. 11:1067–1075, 1997). Searching programs that can be used in this process exist on the Internet, such as Lutefisk 97 (Internet site: www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, Peptide Search and ProFound programs described above. Therefore, adding the sequence of a gene and its predicted protein sequence and peptide fragments to a sequence database can aid in the identification of unknown proteins using tandem mass spectrometry.
Thus, there also exists a need in the art for polypeptides suitable for use in peptide fragmentation studies, for use in molecular weight measurements, and for use in protein sequencing using tandem mass spectrometry.